Phosphoenolpyruvate carboxykinase (PEPCK) has been found by Lardy to be low in Sudden Infant Death Syndrome, upon autopsy, and to be abnormal catalytically. Preliminary data from this laboratory have shown that bromopyruvate inactivates the human liver mitochodria enzyme by a process that is active-site related, with likely sulfhydryl modification. It is proposed to develop a procedure for purifying the enzyme from small amounts of human liver, through application of affinity chromatography, and then examine SIDS and non-SIDS enzyme from mitochondria and cytosol to establish the nature of this inactivation by bromopyruvate with respect to relative rates of inactivation, amino acid residue(s) modified, and substrate protection effects in each instance. Then to be similarly studied are the effects of selected protein modification agents on liver PEPCK activity and structure, with comparisons of reactivity of SIDS to controls. Pig liver enzyme will be used for preliminary studies. Finally, the overall, and active-site, conformations of the enzyme will be studied from computer analysis of the far-ultraviolet region of the circular dichroism spectrum, of unmodified and selectively modified enzyme. The protective effects of salts (against bromopyruvate inactivitation) will be further studied, and analyzed in the visible CD region. Fluorescent probes and reporter groups will be used to probe for hydrophobic and cationic regions at the active-site that may be present and alter sulfhydryl properties at the active-site.